Volume 7 Number 31979

Nucleic Acids Research

Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucel

M.Lheureux*, Martine Lheureux*, T.Vervoort+, N.Van Meirvenne+ and M.Steinert*

*DNpartement de Biologie Moleculaire, Universite libre de Bruxelles, 67, rue des Chevaux, 1640 Rhode-St-Genbse, and Institute of Tropical Medicine, 155, Nationalestraat, 2000-Antwerpen, Belgium Received 3 August 1979

ABSTRACT.

Polyadenylated RNA isolated from total polyribosomes of two variable antigen types (VATs) of T. brucei brucei were shown to program the synthesis, in mRNA-dependant reticulocyte lysates, of a wide variety of polypeptides. After immunoprecipitation of these cell-free products with an homologous antiserum raised against purified variant-specific surface antigen (VSSA), a major electrophoretic band was apparent on fluorography. It was confirmed that this band corresponds to the variable antigen since only an excess of purified homologous antigen will provoke competition. The apparent molecular weight of the in vitro synthesized antigen is The VSSA mRNA has been found in membrane-bound about 63,000 daltons. polyribosomes and a 15 fold immunological purification of this mRNA has been obtained, using partially purified anti-VSSA IgG in conjunction with inactivated Staphylococcus aureus.

INTRODUCTION. Trypanosomes challenge the immune response of their mammalian host by repeatedly changing their antigenic character. Although antigenic variation of trypanosomes is known since the early years of this century (1), it has been so far an insurmountable obstacle to vaccination against trypanosomiases. Progresses in protein chemistry and the development of molecular biology lead however to reniewed hopes for a successful immunoprophylaxis which materialized in a flourishing interest for the surface antigen of trypanosomes and have been expressed in recent reviews of the subject (2-5). In chronic infections successive waves of parasitaemia are generally observed, each new wave being produced by distinct variable antigen types (VATs). It has been established that a single clone of trypanosomes may reversibly express a repertoire of up to about 100 different VATs (6,7), individual cells expressing only one single VAT at a time. Early clonal infections are characterized by serologically nearly homogeneous populations and by subsequent appearance of a repertoire-specific series of predominants VATs (4). According to some authors (4), the same "basic" VATs are C Information Retrieval Limited 1 Falconber Court London Wl V 5FG England

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Nucleic Acids Research always reexpressed first after cyclic transmission of a given clone by the tse-tse fly. The serological specificity of trypanosomes is determined by a coat (8) of variant-specific surface antigen (VSSA) which probably covers their surface so completely that it makes all other potentially immunogenic components of the parasite inaccessible to circulating antibodies. VSSAs are glycoproteins which comprise a single polypeptide chain of about 600 amino acid residues (9). The facts that most or perhaps all the antigenic determinants seem to be associated with this protein moiety, that the amino acid composition and sequence differ widely between the different variant proteins of a single clone (9, 10) and the recurrence of antigenic types after cyclic transmission indicate that antigenic variation is mediated by the controlled expression of individual variant-specific genes and not by mutations. Research on antigenic variation in trypanosomes might be rewarding in at least two ways. Inexpensive production of pure VSSAs in bacterial systems, thanks to the recent development of genetic engineering could pave the way to efficient immunoprophylaxis. On the other hand, a thorough investigation of the regulatory processes involved in the expression of the alternative surface antigens could reveal some vulnerable point to be specifically attacked. Either of these approaches require the isolation of VSSA messenger RNA. Total RNA (11) and poly(A)+RNA (11, 12) from trypanosomes have been recently prepared and shown to induce in reticulocyte or wheat germ in vitro systems the synthesis of proteins which react with homologous VSSAdirected immunoglobins. Partial purification of VSSA mRNA has been obtained by gel electrophoresis (12). In the present study, the variable antigen has been unambiguously identified among the in vitro translation products of poly(A)+RNA prepared from two different variant populations of the same clone of T. brucei brucei. A significant purification of these specific VSSA messengers was carried out after immunoprecipitation of VSSA - synthesizing polyribosomes and we obtained evidence that in vivo these polyribosomes are attached to the membranes of the endoplasmic reticulum.

MATERIALS AND METHODS.

a)

Standard buffers. 2.5 mM NaH2PO4, Phosphate saline glucose buffer (PSG): 47.5 mM Na2O4H 1.5 % TMK 50 mM Tris-HCI buffer: 36.5 mM NaCl, glucose (pH 8.0); , 5 mM MgCGl2, 25 mM KCI (pH 7.5); Polysome immunoprecipitation buffer (PIP): 25 mM Tris-HCI, 150 mM NaCl, 5 mM MgCl2 , 0.5 % Triton X100 , 0.5 % Na deoxycholate (pH 7.5) Immunoprecipitation buffer (IP) : 8 mM Na2HPO4 , 0.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCI, I % Triton XIOO, 0.5 % Na deoxycholate, 1 % dl-methionine, 0.02 %

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Nucleic Acids Research sodium azide (pH 7.2); Tris-SDS buffer 10 mM Tris-HCI, 0.5 % SDS (pH 7.5); Sample electrophoresis buffer (SEB): 80 mM Tris-HCI , 4 % SDS , 5 % 2-mercaptoethanol, 0.25 M sucrose, 10 % glycerol (pH 7.8).

b)

Trypanosomes, growth and harvesting. Clones of two VATs of Trypanosoma brucei brucei, AnTat I and AnTat 8, were isolated from a syringe-passaged line derived from stabilate EATRO 1125 (6). For cryopreservation in liquid nitrogen, infected mouse blood was mixed to an equal volume of Alsever's solution and 10 % glycerol was added as cryoprotectant. Rats were infected intraperitoneally with about 5 X 107 trypanosomes and exsanguinated by cardiac puncture 72 hours later. The blood was collected on heparin as anticoagulant and immediatelly chilled on ice. Unless otherwise stated, all subsequent operations were performed at a temperature comprised between 0 and 4°C. Trypanosomes were separated according to Lanham (13) by ion-exchange chromatography on DEAE-cellulose in PSG buffer. Cycloheximide (10 1pg/ml) was added to this buffer if trypanosomes were intended for the isolation of polyribosomes (see results). The parasites were washed by repeated sedimentations in PSG buffer for 5 min at 2,500 g. The yield was about 5 X 108 trypanosomes per ml of highly infected blood.

c)

Preparation of glycoproteinic antigens. Washed trypanosome- were resuspended in 0.017 M NaCl and in the X-Press cell (LKB, Sweden). The homogenate was centrifuged disintegrated at 40,000 g for 1 hour. The supernatant was dialysed against 0.015 M phosphate buffer pH 8 and passed through a DEAE-cellulose column equilibrated with the same buffer. The unretarded peak which contained mainly variable glycoproteinic antigen was dialysed against 0.02 M phosphate buffer pH 7 containing I M NaCI and further purified by affinity chromatography on Con A-Sepharose (Pharmacia, Sweden). The glycoproteinic fraction was extensively dialysed against distilled water and freezedried. The purification of the clone specific glycoproteinic antigens will be described in detail elsewhere (14) .

d)

Antisera preparation. Antisera against each of the purified glycoproteins were raised in rabbits by subcutaneous inoculation of 1 mg of antigen made up in Freund's complete adjuvant, followed on day 51 by a subcutaneous booster inoculation of I mg of antigen in saline. Sera were collected on day 97 and were stored at -70°C until used. Crude preparations of IgG were obtained by molecular sieving of the sera on Sephadex G 200 (Pharmacia, Sweden) (15) and reconcentrated by ultrafiltration. For 597

Nucleic Acids Research the precipitation of polyribosomes, purer and chiefly ribonuclease-free IgG were prepared by two ammonium sulfate precipitations (16) followed by a combined chromatography on DEAE- and CM-cellulose (17). All IgG preparations were stored at -200C.

e)

Bacterial adsorbent. Inactivated Staphylococcus aureus, strain Cowan I, which were prepared according to Kessler (18), were kindly supplied by Dr E. Hannecart-Pokorni (Institut Pasteur du Brabant). They were stored for several months at 4°C as a 10 % (w/w) suspension in IP or PIP buffers and were repeatedly washed just before being used.

f)

Preparation of total polyribosomes.

Washed cells of T. brucei were suspended in ten volumes of TMK buffer. After the addition of Triton XIOO and Na deoxycholate each to a final concentration of 1 %, lysis was achieved in a glass Dounce homogenizer by 3 strokes of a loose-fitting pestel B and 7 strokes of a tight-fitting pestel A. The nuclei, flagella and other large debris were removed by centrifugation for 5 min at 12,000 g. Total polyribosomes were collected from the supernatant by pelleting at 325,000 g for 45 min through two steps of Pellets of sucrose in TMK buffer, respectively 0,5 M (1 ml) and 2 M (2 ml). were TMK buffer and either polyribosomes cautiously washed by 3 drops of ice-cold resuspended for immediate use or stored in liquid nitrogen. In recent experiments, especially when the polyribosomes were to be subsequently submitted to immlune precipitation, the polyribosomes were not pelleted but harvested at the boundary of two sucrose layers of different densities: pelleting was expected to favor aggregation of cosedimenting polyribosomes, entailing a contamination of the immune precipitated polyribosomes. Indeed, a substantial reduction of contamination could be obtained by collecting the polyribosomes by floatation. Thus sedimentation was performed in a SW 41 rotor at 260,000 g for 65 min through two layers of 0.5 M (0.5 ml) and I M (3.5 ml) sucrose in TMK buffer onto a cushion of 2.5 M sucrose (1.5 ml). The opalescent polyribosome band was collected by puncturing the side of the tube with a sterile syringe just under its lower boundary. Qualitative analysis of polyribosomes was performed by sedimentation through a 12.5 ml linear 20 % - 50 % (w/v) sucrose gradient for 60 min at 260,000 g. Following centrifugation, the absorbance at 254 nm of the content of the tube was monitored continuously by a Cary 15 spectrophotometer.

g)

Isolation of free and membrane-bound polyribosomes. The cells, to which no detergent was added , were lysed in TMK buffer by a simple passage in a French press (5,400 psi). Cellular fragments were eliminated by 598

Nucleic Acids Research centrifugation at 12,000 g for 5 minutes. The microsomes were then pelleted at 30,000 g for 20 min and free polyribosomes recovered from the supernatant as described above. Washed microsomes were resuspended in TMK buffer containing I % of both Triton XIOO and Na deoxycholate and dispersed by 5 strokes of a tight -fitting pestel A. The sample was then centrifuged at 30,000 g for 20 min and the membranebound polyribosomes isolated from the supernatant as described above for total polyribosomes.

Immunoprecipitation of AnTat I variant-specific polyribosomes. Total purified polyribosomes prepared by banding, were dialysed against polyribosome immunoprecipitation (PIP) buffer. Before precipitation, they were dispersed in a Dounce homogenizer. Polyribosomes and purified IgG were mixed in order to obtain final concentrations of, respectively, 10 A260 units/ml and 0.5 mg/ml. After incubating for 2 1/2 hours at 0° C, phenylmethylsulfonyl fluoride (PMSF) was added (1 mM final concentration) and the mixture received I g inactivated Staphylococcus aureus for each 16 mg IgG. If required,the sample was diluted with PIP buffer in order not to exceed a final bacterial concentration of 2 % (w/v). Incubation was resumed for an additional 1S min at 00 C. The precipitated complexes were separated by centrifugation through a discontinuous gradient of 0.5 M - I M sucrose in PIP buffer adjusted to 0.5 M NaCl and then repeatedly washed in PIP buffer. The precipitates were finally resuspended in ten volumes of Tris-SDS buffer and incubated for a few minutes at room temperature so as to cause the discharge of adsorbed polyribosomes from the bacteria, which were then spun down (5 min at 8,000 g). The treatment also dissociate the polyribosomes. Non-adsorbed polyribosomes were recovered from the first supernatant, concentrated by pelleting and dissociated in Tris-SDS buffer. h)

i)

Isolation of messenger RNA. The polyribosomes were dissociated in Tris-SDS buffer at a concentration of about 10 A260 units/ml. Proteinase K (E. Merck, Darmstad) ( I mg/ml) was autodigested in the same buffer for 15 min at 250 C immediately before being used and added to the sample at the final concentration of lOO1pg/ml (19). Following incubation (30 min at 370 C), the digest was heated for 5 min at 80° C, then cooled in an ice-water bath and adjusted to 0.5 M NaCl. Oligo(dT)-cellulose (P-L Biochemicals, Inc., Milwaukee, Wis.) chromatography was carried out at room temperature as detailed by Arnemann et al. (20). Briefly, poly(A)+RNA was bound by three passages of the solution through the column. The gel was extensively washed with the same buffer (Tris-SDS + 0.5 M NaCI), then with this buffer deprived of SDS. Bound RNAs were eluted with sterile distilled 599

Nucleic Acids Research water and recovered by precipitation with two volumes of ethanol in the presence of 0.25 M NaCI. The precipitate was allowed to form overnight at -20° C.

Translation of mRNA in a cell-free rabbit reticulocyte system. Rabbits weighing about 3.5 kg were made anaemic by four daily subcutaneous injections of 2.5 % (w/v) solution of phenylhydrazinium chloride in 0.9 % NaCl (0.25 ml per kg). They were bled on the 6th day by cardiac puncture under Nembutal anaesthesia, clotting being prevented by the addition of heparin (2,500 units). The reticulocytes were collected by low speed centrifugation (10 min at 600 g), washed three times with 0.9 % NaCI, then lysed by gentle stirring for 2 min in one volume of ice-cold distilled water. The debris and white cells were removed by centrifugation at 12,000 g for 20 min at 00 C. The supernatant was divided in 0.8 ml aliquots and stored under liquid nitrogen. The reticulocyte system was made "mRNA-dependant" and supplemented according to Pelham & Jackson (21), except that 25 pg/ml of micrococcal in our hands this enzyme nuclease (EC.3.1.4.7) were used for pretreatment: concentration gave the best added mRNA-specific response (data not shown). About 100 pCi of [35S] methionine (approx. 106 Ci/mol) (The Radiochemical Centre, Amersham) was added in I ml of lysate as well as the 19 unlabeled amino acids at concentrations related to their mean frequency of occurence in the few VSSAs of T. brucei that have been so far analysed (2). Routinely, 20 pg/ml of mRNA were introduced in this cell-free translation Total incorporation was measured as described (21), but acid-precipitates were system. heated for 20 min at 800 C in order to unload the tRNAs. To screen protein synthesis, 3 pl aliquots of the total system were submitted to polyacrylamide gel electrophoresis. j)

k)

Immunoprecipitation of the cell-free translation products. Immunoprecipitation of in vitro synthesized polypeptides was performed in a final sample volume of 450 p1l, diluting aliquots of the lysate twenty to fiftyfold in immunoprecipitation (IP) buffer. Each sample received 3 p1 of crude IgG. In competition control experiments, 20 pg of purified VSSA or the same amount of ovalbumin were added just before mixing with the immunoglobulins. Incubation was carried out for 2 hours at 370 C and then overnight at 40 C (22). Thereafter the samples were adjusted to 1 mM PMSF and 60 p1i of a 10 % (w/w) suspension of inactived S. aureus were added. Incubation was continued for 4 hours at 40 C, with discontinuous agitation. The precipitated complexes were washed twice by 10 min centrifugations in a Beckman microcentrifuge through a 0.5 ml cushion of I M sucrose in IP buffer (adjusted to 0.5 M NaCl), and further washed by a 5 min spin in this buffer. 600

Nucleic Acids Research 1)

SDS polyacrylamide gel electrophoresis and fluorography. Total cell-free synthesized products or their immune precipitated fractions were analysed in SDS 15 % polyacrylamide gels, using a vertical slab gel apparatus with the discontinuous buffer system of King and Laemmli (23) and monomers recrystallized according to Loening (24). The samples were either diluted or dissolved in 50 p1 of sample electrophoresis buffer (SEB) containing 0.02 % bromophenol blue, boiled for 3 min and briefly centrifuged in order to eliminate aggregates and/or bacterial walls before applying onto the gel. Electrophoresis was carried out for 2 1/2 hours at 40 mA/plate at room temperature. After staining with Coomassie Brillant Blue and destaining, the gel was processed for 2 1/2 hours with 22.2 % (w/v) 2,5-diphenyloxazole (PPO) in dimethylsulfoxide (DMSO) and dried on Whatman 3MM filter paper. The radioactive bands were detected by fluorography (25), on presensitized Kodak XRP-1 plates , with Kodak X-0- matic regular salts intensifying screens.

RESULTS. In addition to our efforts to keep the mRNAs intact, we have tried to maintain the largest number of ribosomes attached to the polyribosomes in order to favor immunological purification of a peculiar class of mRNA by reaction with the nascent polypeptide citains. The most significant improvement was achieved by adding cycloheximide to the PSG buffer of the DEAE-cellulose column (Fig.l), the ratio LP/T (36) beeing increased from 42 % to 65 % in presence of 10 pg/ml of this inhibitor. On the contrary, its presence in the TMK buffer used for cell disruption modified only

0.3-

0.2 -0.2-

0.1

0.1

A

vvB

v

Figure 1. Sucrose gradient analyses of polyribosomes prepared from T.brucei. Trypanosomes were separated from the blood cells in absence (A) or presence (B) of cycloheximide (10 pg/ml). 601

Nucleic Acids Research very slightly the size distribution of the polyribosomes (data not shown). About 0.025 A260 units of poly(A)+ RNA was extracted from each A260 unit of total trypanosome polyribosomes. These poly(A)+ RNAs were tested in a rabbit reticulocyte lysate and SDS polyacrylamide gel electrophoresis was used to screen total polypeptide synthesis as well as peptides precipitated by various IgG preparations. Figures 2 and 3 illustrate the in vitro translation of poly(A)+ RNAs extracted from serotype AnTat 1; essentially similar results being obtained with poly(A)+ RNAs extracted from serotype AnTat 8 (not shown). A broad spectrum of polypeptides varying in size from about 20,000 to 90,000 daltons was produced (Fig.2, lane C and Fig.3), with a serie of major bands appearing in the range of 5 to 6 x 10 daltons. The comparison of lanes D to I of Fig.2 allowed us to ascribe with certainty the major electrophoretic band (arrowed) obtained after immunoprecipitation of the products of in vitro translation to the surface antigen of serotype AnTat 1. Indeed, this band is not revealed after immunoprecipitation with IgG from non-immune

A

Figure 2.

B

C

D

E

FE CG

H

Electrophoretic and fluorographic analyses of the polypetides synthesized in vitro in a rabbit reticulocyte lysate without addition of exogenous template (lane A), with purified from rabbit 9S globin mRNA (B), and in the presence of total poly(A)+ RNA T. brucei (C). The products of total poly(A)+ RNA translation (as shown in lane C) have been submitted to different immune precipitation tests. The complexes, analysed in lanes D to I, have been obtained by reaction with IgG from a non-immune serum (D), heterologous anti-AnTat 8 IgG (E), homologous anti-AnTat 1 IgG (F and G), anti-AnTat 1 IgG in the presence of an excess of cold purified AnTat 1 antigen (H), anti-AnTat 1 IgG as in H but in the presence of ovalbumin instead of the cold antigen (I). The arrow points to the band identified as the AnTat I variant-specific surface antigen. 602

Nucleic Acids Research

Figure 3.

SDS-polyacrylamide gel electrophoresis of the in vitro translation products of total T. brucei AnTat I poly(A)+RNA (continuous densitometric tracing) and of the fraction of these products precipitated by homologous anti-AnTat 1 IgG (dotted line). The polypeptides used as reference in adjacent lanes are bovine serum albumin (BSA), ovalbumin (OA) and soybean trypsin inhibitor (STI) (67,000, 43,000 and 20,100 daltons respectively). Simple arrows point to polypeptides made on residual rabbit mRNA. The precipitated variant-specific polypeptide is marked by the double arrow. serum (D) or with an heterologous IgG preparation (E). Furthermore the addition of an excess of unlabelled purified homologous antigen inhibits its precipitation (H) and this competition is not observed when ovalbumin is added instead of the unlabelled VSSA (I). It should be noted that among the abundant in vitro translation products of total poly(A) containing RNA, surprisingly little labelling was found at the presumed VSSA site in the absence of immunoorecipitation (Fig.2, lane C and Fig.3). Some of the electrophoretic bands are rabbit proteins, since they also appear in the absence of any trypanosome mRNA (Fig.3). Minor bands (40,000 to 60,000 d) observed in immune precipitates obtained with heterologous as well as 603

Nucleic Acids Research homologous IgG does not seem to represent antigenic determinants specifically recognized by the immune sera, as they also appear with normal serum. However, we found that the relative intensity of these labelled bands decreased slightly when unlabelled VSSA, but not ovalbumin, was added. The template activity of T. brucei poly(A)+ RNA isolated from either free or membrane-bound polyribosomes were compared by testing in rabbit reticulocyte systems. The distribution of in vitro synthesized polypeptides was similar for the two preparations of mRNAs, except in the region of high molecular weight products, which were made more abundantly from membrane-associated mRNAs(Fig.4, C and D). After immunoprecipitation (lanes E and F) performed on aliquots containing the same amount of trichloracetic acid-precipitable radioactivity, it became clear that most of the VSSA specific mRNA was in the microsome fraction. The presence of some VSSA mRNA in the free polyribosome fraction may reflect a possible discharge of polyribosomes from microsome membranes, due to the particularly drastic cell disruption conditions which had to be used. Effective immunological precipitation of polyribosomes which synthesize a A

B

C

D

E

F

Figure 4. Electro?phoretic and fluorographic analyses of the in vitro translation products of poly(A) RNA prepared from free (lane C) and membrane-boun-d (D) polyribosomes of

T. brucei AnTat 1. Controls show the residual translation in the reticulocyte lysate in the absence of exogenous template (B) and the translation of added purified 95 rabbit globin mRNA (A). Lanes E and F contain fractions of the translation products analyzed in lanes C and D respectively, which have been precipitated by homologous antiAnTat I lgG. The arrow points to the VSSA polypeptide. 604

Nucleic Acids Research given protein requires the presence of specific antigenic determinants on the nascent polypeptide chain. It is thus fortunate that most if not all the specific determinants of VSSA belong to its protein moiety (see discussion) and, in addition, immunodiffusion tests have shown that still uncompleted polypeptide chains of VSSA are effectively and specifically recognized by homologous IgG (Fig.5). It was observed that the specificity of the immune precipitation of VSSA specific polyribosomes could be improved if the total polyribosome fraction had been first harvested by floatation instead of pelleting (see Materials and Methods). Thus most of the experiments referred to here have been made with polyribosomes obtained in this way. Two kinds of preparations were carried out on freshly isolated polyribosomes. Polyribosomes were directly processed by the proteinase K and oligo (dT)-cellulose technique to give total polyribosomal poly(A)+RNA; altematively they were first submitted to immunoprecipitation with homologous or heterologous IgG. Poly(A)+RNA were then extracted from the precipitated and non-precipitated polyribosome fractions in the same way as from total polyribosomes. The recovery of poly(A)+RNA from the sample submitted to immune precipitation was better than 90 % and about 6.5 % of these RNA were found in the precipitated immune complex. Their messenger activity was studied in the rabbit reticulocyte cell-free system as illustrated in Figure 6 for an experiment perf4rmed with serotype AnTat 1. Similar results have been obtained with serotype AnTat 8 (data not shown). While the polypeptide corresponding to the VSSA is barely visible after stimulation with total polyribosomal poly(A)+RNA (lane B), the corresponding band appears very clearly when the acellular system was set up by mRNA from the immune precipitated polyribosomes (lane C). This enrichment in VSSA mRNA is still more obvious by the comparison of the VSSA bands in

Figure 5. Immunodiffusion reaction between variant-specific IgG and the nascent polypeptide chains released from total trypanosome polyribosomes by 0.1 % SDS. Contents of the wells are: 1) anti-AnTat 1 IgG, 2) anti-AnTat 8 IgG, 3) polyribosomes from serotype 5) purified AnTat I antigen, AnTat 1, 4) polyribosomes from serotype AnTat 8, 6) purified AnTat 8 antigen. 605

Nucleic Acids Research A B

C D E F G

H

I

J K

l M

N O

P

Figure 6. Analyses of the in vitro translation products of total T. brucei AnTat I polyribosomal poly(A)+RNA (lane B,of poly(A) RNA derived from polyribosomes precipitated with homologous anti-AnTat I IgG (lanes C,H- and L), poly(A) RNA derived from polyribosomes precipitated with heterologous anti-AnTat 8 IgG (lane I) and of poly(A)+RNA prepared from the polyribosomes which remained in the supernatants of immune precipitations with anti-AnTat 1 IgG (lane N). Lanes A and P contain the products of the in vitro residual translation in the absence of exogenous template. In lanes D and E are the products of total poly(A)+RNA translation which have been immune precipitated with homologous anti-AnTat I IgG in the absence and in the presence of an excess of cold AnTat I antigen, respectively. Lanes F and G show similarly the translation products of poly(A)+RNA derived from immune precipitated polyribosomes, after precipitation with anti-AnTat I IgG in the absence (lane F) and presence (lane G) of an excess of cold AnTat 1 antigen. Lanes 3, K, M and 0 contain fractions of the translation products analysed in lanes H, I, L and N respectively, which have been precipitated by homologous anti-AnTat I IgG. Arrow points to the VSSA polypeptide chain. lanes D and F, where the in vitro translation products have been immune precipitated from aliquots containing the same amount of trichioracetic acid-precipitable radioactivity. The VAT-specificity of the polyribosome immunoprecipitation is evident (lanes H to K), since no VSSA could be detected among the translation products of poly(A)+RNA isolated from polyribosomes which have been precipitated by heterologous lgG (I), even if these products were f urther submitted to immune precipitation with homologous IgG to provoke its concentration (K). Moreover, the ef ficiency of the polyribosome selection is demonstrated by the comparison of the in vitro VSSA production after stimulation by RNA poly (A)+ isolated from precipitated of nonprecipitated polyribosome fractions (lanes L, M versus N, 0).

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Nucleic Acids Research DISCUSSION. Polyribosomes have been isolated from variants AnTat I and AnTat 8 of the same clone, itself derived from stabilate EATRO 1125 of Trypanosoma brucei brucei. As shown by analytical gradient centrifugation the major part of the preparation is consisted of polyribosomes of more than 8 ribosomal units, indicating little or no degradation. Polyadenylated RNAs were prepared from these polyribosomes and translated in a cell-free reticulocyte system. The labelled products of this in vitro translation are highly heterodisperse, most of them having a size comprised between 2 x 104 and 9 X 104 daltons, with a series of major bands in the range of 5 to 6 X 104 daltons, thus close to the size expected for the coat antigen protein. By testing the products of this in vitro translation with the specific IgG raised against the homologous and the heterologous VSSA, we could unambiguously identify the variant-specific surface antigen as a polypeptide of about 6.3 X 104 daltons. This size is slightly larger than the one measured by others (11, 12), the difference being due, most probably, to the use of distinct T. brucei strains and variants. Nevertheless, this size is well the one expected for the complete protein moiety of a VSSA. The finding that the VSSA produced in vitro reacts specifically with the homologous IgG is in agreement with the observations of others (11, 12) and strongly supports the view that variant specific determinants are carried by the protein rather than by the glycane part of the antigen. It is indeed highly improbable that glycosylation, a function performed in vivo by the Golgi complex and by the endoplasmic reticulum, would occur in the reticulocyte in vitro system. Most of the messengers coding for the VSSA were found in membrane bound polyribosomes. This observation was to be expected from a protein assigned to the cell surface and it fits also with observations of an increased development of the endoplasmic reticulum and of the Golgi apparatus in the bloodstream form, as compared to the culture or vector forms which lack the coat (26). A partial purification of VSSA messenger RNA has been obtained by gel preparative Williams et al. (12) from total polyribosomal poly(A)+ RNA by electrophoresis. A draw back of this method in the present case is that it selects according to molecular size, whereas the VSSA messenger RNA belongs to precisely the most abundant size class of poly(A)+RNA as judged from the in vitro translation tests of poly(A)+ RNA fractions obtained by preparative gel electrophoresis (12) or sucrose gradient centrifugation (experiment not shown). Moreover, the trypanosomes are very rich in microtubules and tubuline mRNAs, which are expected to be of approximately the same size as VSSA messengers, could be major contaminants. -

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Nucleic Acids Research Immunoprecipitation has been first suggested as a general procedure for isolating specific polyribosomes by Cowie et al. (27). After experimenting different immunological techniques, we settled for the use of Staphylococcus aureus cells to collect the immune complexes. The methods, as described in Materials and Methods, has been rather extensively modified from Mueller-Lantzsch and Hung Fan (28). Applied to variants AnTat I and AnTat 8 of T. brucei, a fraction of polyribosome was isolated which contained about 6.5 % of total polyribosomal poly(A)+ RNA. Since this fraction includes most if not all the VSSA specific mRNA, as demonstrated by the in vitro translation tests, it is concluded that this mRNA has been purified about 15 fold. As the coat protein represents about 10 % of total cell proteins in found somewhat surprising that so little VSSA could be detected among we T. brucei (2), the products of in vitro translation of total polyribosomal poly(A)+ RNA (Figs.2 and 3). There are several possible explanations for this observation: 1) the VSSA specific mRNA is not abundant but its translation is highly efficient in vivo. 2) the messenger is relatively abundant and shows little efficiency in the heterologous rabbit - derived in vitro translation system. It has indeed been established that selection of mRNA for translation may occur at the initiation level: this has been found in vivo both in homologous (29) and heterologous living cells (30, 31), as well as in cellfree systems derived from wheat germ (32), from Krebs II ascites cells (33, 34) and from rabbit reticulocytes (35). 3) our poly(A)+ RNA extraction procedure was counterselective with regard to VSSA messengers. 4) autoradiographic estimation of the labelled VSSA produced in vitro is low owing to its relatively low content in methionine

(9). To summarize, the messenger RNA for two variant - specific surface antigens of T. brucei brucei have been isolated and characterized by in vitro translation in rabbit reticulocyte lysates. The VSSA mRNA has been found in membrane-bound polyribosomes and a 15 fold purification of this mRNA has been obtained by immune precipitation.

ACKNOWLEDGMENTS. This investigation received support from the Institut National de la Sante et de la Recherche Medicale (I.N.S.E.R.M., Paris), of the Fonds de la Recherche Scientifique Medicale (F.R.S.M., Brussels) and of the World Health Organization (W.H.O., Geneva). M.L. holds a fellowship of the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (I.R.S.I.A., Brussels). This support is gratefully acknowledged. We would like also to 608

Nucleic Acids Research express our gratitude to Drs. G. Marbaix, G. Huez and J. Ghysdael for helpful discussions and for their interest in this work.

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